T cells in solid tumors : investigating the immunomodulation in the tumor microenvironment

The immune system protects human from cancer through an immunosurveillance mechanism. However, the progressive nature of tumor cells to differentiate and the complexity of the tumor microenvironment may result in the immunomodulation of immune cells. In this thesis, we aim to explore the T cell immunomodulation inside the intricate solid tumor microenvironment in patients. First, we investigated suppressive regulatory T cells (Tregs) in urinary bladder cancer (UBC). Our group previously demonstrated a contradictory finding that a high FOXP3+ tumor infiltrating lymphocyte (TIL) number correlates positively to survival. In here, we answered that FOXP3+ CD4+ T cells in the tumor were real Tregs which protectively regulated tumor invasiveness by suppressing MMP2 expression in tumor-associated macrophages (TAMs) and tumor cells. Next, we explored the subset of tissue-resident memory CD8+ T (TRM) cells from UBC tumor. It is less known whether TRM cells are effective killers of tumor cells. We revealed that tumor TRM cells were epigenetically committed to express perforin. Although TRM cells expressed exhaustion marker PD-1, they were not terminally exhausted. As a result, we found that an increased number of TRM cells in the tumor correlated with a lower tumor stage. Furthermore, we looked into the cytotoxic CD8+ T cells in the sentinel nodes (SNs) of UBC patients. Surprisingly, we discovered that SN CD8+ T cells displayed a deficiency of their cytotoxic constituent perforin, whereas granzyme B was still expressed. Thereafter, we revealed that muscle invasive UBC suppressed perforin expression using an ICAM-1/TGFβ2 – mediated pathway as an immune escape mechanism. In the next study, we focused on the effect of standard neoadjuvant chemotherapy (NAC) and T cell responses in the SNs. We found that NAC reinforced the anti-tumor T cell activities by reducing the exhaustion in CD8+ and CD4+ effector T cells, which consequently increased their cytotoxicity and clonal expansion, respectively. Additionally, NAC also reduced the frequency and activation of the suppressive Tregs. Lastly, as a result of escaping the immune destruction, tumor can grow and metastasize. In this study, we revealed that micrometastases in lymph nodes of renal tumors could be reliably detected by flow cytometry. This method is more sensitive, objective, time- and cost-effective compared to the gold standard histopathological examination. In conclusion, T cells are modulated in the solid tumor microenvironment. By understanding the molecular and cellular aspects of T cells in this microenvironment, we may unveil new strategies for designing cancer immunotherapies in the future

IL-15-dependent immune crosstalk between natural killer cells and dendritic cells in HIV-1 elite controllers

Summary: As the principal effector cell population of the innate immune system, natural killer (NK) cells may make critical contributions to natural, immune-mediated control of HIV-1 replication. Using genome-wide assessments of activating and inhibitory chromatin features, we demonstrate here that cytotoxic NK (cNK) cells from elite controllers (ECs) display elevated activating histone modifications at the interleukin 2 (IL-2)/IL-15 receptor β chain and the BCL2 gene loci. These histone changes translate into increased responsiveness of cNK cells to paracrine IL-15 secretion, which coincides with higher levels of IL-15 transcription by myeloid dendritic cells in ECs. The distinct immune crosstalk between these innate immune cell populations results in improved IL-15-dependent cNK cell survival and cytotoxicity, paired with a metabolic profile biased toward IL-15-mediated glycolytic activities. Together, these results suggest that cNK cells from ECs display a programmed IL-15 response signature and support the emerging role of innate immune pathways in natural, drug-free control of HIV-1

Facing the future : challenges and opportunities in adoptive T cell therapy in cancer

INTRODUCTION: In recent years, immunotherapy for the treatment of solid cancer has emerged as a promising therapeutic alternative. Adoptive cell therapy (ACT), especially T cell-based, has been found to cause tumor regression and even cure in a percentage of treated patients. Checkpoint inhibitors further underscore the potential of the T cell compartment in the treatment of cancer. Not all patients respond to these treatments; however, many challenges remain. AREAS COVERED: This review covers the challenges and progress in tumor antigen target identification and selection, and cell product manufacturing for T cell ACT. Tumor immune escape mechanisms and strategies to overcome those in the context of T cell ACT are also discussed. EXPERT OPINION: The immunotherapy toolbox is rapidly expanding and improving, and the future promises further breakthroughs in the T cell ACT field. The heterogeneity of the tumor microenvironment and the multiplicity of tumor immune escape mechanisms pose formidable challenges to successful T cell immunotherapy in solid tumors, however. Individualized approaches and strategies combining treatments targeting different immunotherapeutic aspects will be needed in order to expand the applicability and improve the response rates in future

Urothelial bladder cancer may suppress perforin expression in CD8+ T cells by an ICAM-1/TGFβ2 mediated pathway

The immune system plays a significant role in urothelial bladder cancer (UBC) progression, with CD8+ T cells being capable to directly kill tumor cells using perforin and granzymes. However, tumors avoid immune recognition by escape mechanisms. In this study, we aim to demonstrate tumor immune escape mechanisms that suppress CD8+ T cells cytotoxicity. 42 patients diagnosed with UBC were recruited. CD8+ T cells from peripheral blood (PB), sentinel nodes (SN), and tumor were analyzed in steady state and in vitro-stimulated conditions by flow cytometry, RT-qPCR, and ELISA. Mass spectrometry (MS) was used for identification of proteins from UBC cell line culture supernatants. Perforin was surprisingly found to be low in CD8+ T cells from SN, marked by 1.8-fold decrease of PRF1 expression, with maintained expression of granzyme B. The majority of perforin-deficient CD8+ T cells are effector memory T (TEM) cells with exhausted Tc2 cell phenotype, judged by the presence of PD-1 and GATA-3. Consequently, perforin-deficient CD8+ T cells from SN are low in T-bet expression. Supernatant from muscle invasive UBC induces perforin deficiency, a mechanism identified by MS where ICAM-1 and TGFβ2 signaling were causatively validated to decrease perforin expression in vitro. Thus, we demonstrate a novel tumor escape suppressing perforin expression in CD8+ T cells mediated by ICAM-1 and TGFβ2, which can be targeted in combination for cancer immunotherapy

Perforin deficiency in CD8⁺ T cells from sentinel nodes.

<p>(<b>A</b>) The expression of granzyme B and perforin in CD8⁺ T cells of different tissues were phenotyped by flow cytometry. The co-expression pattern in CD8⁺ T cells was shown in dot plots and gated for distinguishing between double and single expression of granzyme B and perforin. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. Dot plots showed a representative data from a patient underwent transurethral resection of the bladder (TUR-B) and cystectomy. (<b>B</b>) The frequency of granzyme B⁺/perforin⁺ CD8⁺ T cells from PBMC, SN, and tumor tissues was shown in graphs and was calculated out of CD8⁺ T cells (n = 27). (<b>C</b>) Same as in (B) but the analysis was done on granzyme B⁺/perforin^- CD8⁺ T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. (<b>D</b>) The expression of gene responsible in encoding granzyme B (<i>GZMB</i>) and perforin (<i>PRF1</i>) in CD8⁺ T cells isolated from PBMC, SN, and tumor (n = 6). RT-qPCR was done to analyze the gene expression followed by quantification using 2^-ΔΔCt method. The fold change was calculated in regards of PBMC as control, with <i>RPII</i> gene used as the housekeeping gene. The data are the means of Log₂ of fold change (2^-ΔΔCt) with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test on each gene. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Tc1 conditions can restore perforin expression in CD8⁺ T cells from sentinel nodes.

<p>CD8⁺ T cells sorted from sentinel node (SN) were cultured in Tc1 conditions <i>in vitro</i> for seven days in order to rescue perforin expression. These SN-derived CD8⁺ T cells were stimulated with anti-CD3 and anti-CD28 stimulating antibodies with the presence of IL-12 and IL-2 cytokines, as well as an anti-IL-4 neutralizing antibody. At the end of the culture, cells were analyzed by flow cytometry and RT-qPCR. (<b>A</b>) Dot plots showed the flow cytometry data from a representative patient for granzyme B vs. perforin expression, before and after the stimulation. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. (<b>B</b>) Flow cytometry result of T-bet expression percentage from CD8⁺ T cells pre- and post-stimulation was analyzed. The frequency of T-bet expression was calculated from CD8⁺ T cells. (<b>C</b>) <i>TBX21</i> and <i>GATA3</i> gene expression analysis was done by RT-qPCR from cells in different culture conditions. <i>RPII</i> gene was used as housekeeping gene and the fold change was calculated based on cells without IL-12 and anti-IL-4 as control using 2^-ΔΔCt method.</p

Sentinel node CD8⁺ T cells with perforin deficiency are exhausted Tc2 cells.

<p>(<b>A</b>) CD8⁺ T cells isolated from sentinel node (SN) were further phenotyped using flow cytometry to demonstrate the difference in T cells exhaustion markers expression (PD-1) and Tc1 transcription factor (T-bet) between granzyme B⁺/perforin⁻CD8⁺ T cells (green box) and granzyme B⁺/perforin⁺ CD8⁺ T cells (red box). The expression of PD-1 and T-bet were shown in dot plots from a representative patient and gated based on isotype control. (<b>B</b>) The frequency of PD-1 and T-bet from (A) was calculated either out of granzyme B⁺/perforin⁻or granzyme B⁺/perforin⁺ CD8⁺ T cells. The data are means with the error bars indicating SEM. Mann-Whitney was used as the statistical test. (<b>C</b>) The expression of T-bet, encoded by <i>TBX21</i> gene, was compared among CD8⁺ T cells sorted from peripheral blood mononuclear cells (PBMC), sentinel node (SN), and tumor. mRNA was extracted from the sorted cells and the <i>TBX21</i> gene expression was analyzed by RT-qPCR. The expression of <i>TBX21</i> was quantified using 2^-ΔΔCt method and the fold change was calculated in regards of PBMC as control. <i>RPII</i> gene was used as housekeeping gene. The data are means with error bars indicating SEM. Kruskal-Wallis was used as the statistical test. (<b>D</b>) Same as in (C), but the analysis was done on <i>GATA3</i> gene expression. (<b>E</b>) The frequency of naïve T cells (CD45RA⁺ CCR7⁺), central memory T (T_CM) cells (CD45RA^- CCR7⁺), effector memory T (T_EM) cells (CD45RA^- CCR7^-), and effector memory T with CD45RA expression (T_EMRA) cells (CD45RA⁺ CCR7^-) was calculated either out of granzyme B⁺/perforin⁻or granzyme B⁺/perforin⁺ CD8⁺ T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

ICAM-1 and TGFβ2 signal from muscle invasive UBC causes perforin downregulation.

<p>Culture supernatants of urothelial bladder cancer (UBC) cell lines were acquired from RT4 (non-muscle invasive) and 5637 (muscle invasive) cell lines. CD8⁺ T cells were then isolated from peripheral blood of healthy donor and cultured in <i>vitro</i> with these supernatants for five days. (<b>A</b>) Analysis of perforin coding gene (<i>PRF1</i>) expression was done by RT-qPCR. mRNA was extracted post-culture from the cells of the culture groups. Bar graphs show different expression of <i>PRF1</i> in CD8⁺ T cells cultured <i>in vitro</i> between RT4 (non-muscle invasive) and 5637 (muscle invasive) supernatant. <i>RPII</i> gene was used as housekeeping gene and the fold change was calculated in regards of RT4 medium using 2^-ΔΔCt method. The data are means with error bars indicating SEM. Paired-t-test was used as the statistical test. (<b>B</b>) Flow cytometry analysis of CD8⁺ T cells at the end of culture was done. The results comparing three groups were shown in dot plots from a representative healthy donor and gated based on isotype control. (<b>C</b>) The frequency of perforin^- CD8⁺ T cells from (B) was counted out of CD8⁺ T cells. The data are means with the error bars indicating SEM. One-way repeated-measure ANOVA was used as the statistical test. (<b>D</b>) Mass spectometry (MS) analysis identified proteins expressed by RT4 and 5637 cell line. Proteins under the category “immune system process” on the GO (Gene Ontology) term were selected for network analysis based on STRING database. Size represented differential expression between RT4 and 5637 supernatants and the color represented betweenness which marked the influence of the protein to the network. Color indicators: blue = low, yellow = average and red = high. (<b>E</b>) The expression of ICAM-1 was validated by flow cytometry on RT4 and 5637 cell line. RT4 and 5637 cells were identified by EpCAM expression. (<b>F</b>) Validation of perforin downregulation by ICAM-1 and TGFβ2 was done <i>in vitro</i> on CD8⁺ T cells isolated from healthy donors in the presence of anti-CD3 stimulating antibody for 5 days. Perforin coding gene (<i>PRF1</i>) expression was done by RT-qPCR. mRNA was extracted post-culture from the cells. Bar graphs show different expression of <i>PRF1</i> in CD8⁺ T cells cultured <i>in vitro</i> between control and TGFβ2 + ICAM-1 + αCD3. RPII gene was used as housekeeping gene and the fold change was calculated in regards of blank medium using 2-ΔΔCt method. The data are means with error bars indicating SEM. Paired-t-test was used as the statistical test. (<b>G</b>) Flow cytometry analysis of CD8⁺ T cells at the end of culture was done. The results were shown in dot plots and gated based on isotype control from a representative healthy donor. The frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. (<b>H</b>) The frequency of granzyme B⁺/perforin^- expressing cells from (G) was counted out of CD8⁺ T cells. The data are means with error bars indicating SEM. Paired-t-test was used as the statistical test. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Production and secretion of perforin in SN CD8⁺ T cells are low after <i>in vitro</i> reactivation.

<p>Lymphocytes isolated from peripheral blood (PBMC) and sentinel node (SN) were cultured for seven days with addition of autologous tumor homogenate. (<b>A</b>) Flow cytometry was done to phenotype the co-expression in CD8⁺ T cells from PBMC and SN before and after reactivation. The results were shown in dot plots and gated based on isotype control. The frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. Dot plots showed data from a representative cystectomized patient. (<b>B</b>) Intracellular perforin was measured by Median Fluorescence Intensity (MFI) post 7-day culture using flow cytometry from (A). The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. (<b>C</b>) The concentrations (pg/ml) of secreted granzyme B and perforin after seven days of culture were analyzed by ELISA and compared between <i>in vitro</i> culture supernatants of PBMC and SN. The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Increased CD4+ T cell lineage commitment determined by CpG methylation correlates with better prognosis in urinary bladder cancer patients

BACKGROUND: Urinary bladder cancer is a common malignancy worldwide. Environmental factors and chronic inflammation are correlated with the disease risk. Diagnosis is performed by transurethral resection of the bladder, and patients with muscle invasive disease preferably proceed to radical cystectomy, with or without neoadjuvant chemotherapy. The anti-tumour immune responses, known to be initiated in the tumour and draining lymph nodes, may play a major role in future treatment strategies. Thus, increasing the knowledge of tumour-associated immunological processes is important. Activated CD4+ T cells differentiate into four main separate lineages: Th1, Th2, Th17 and Treg, and they are recognized by their effector molecules IFN-γ, IL-13, IL-17A, and the transcription factor Foxp3, respectively. We have previously demonstrated signature CpG sites predictive for lineage commitment of these four major CD4+ T cell lineages. Here, we investigate the lineage commitment specifically in tumour, lymph nodes and blood and relate them to the disease stage and response to neoadjuvant chemotherapy. RESULTS: Blood, tumour and regional lymph nodes were obtained from patients at time of transurethral resection of the bladder and at radical cystectomy. Tumour-infiltrating CD4+ lymphocytes were significantly hypomethylated in all four investigated lineage loci compared to CD4+ lymphocytes in lymph nodes and blood (lymph nodes vs tumour-infiltrating lymphocytes: IFNG -4229 bp p &lt; 0.0001, IL13 -11 bp p &lt; 0.05, IL17A -122 bp p &lt; 0.01 and FOXP3 -77 bp p &gt; 0.05). Examination of individual lymph nodes displayed different methylation signatures, suggesting possible correlation with future survival. More advanced post-cystectomy tumour stages correlated significantly with increased methylation at the IFNG -4229 bp locus. Patients with complete response to neoadjuvant chemotherapy displayed significant hypomethylation in CD4+ T cells for all four investigated loci, most prominently in IFNG p &lt; 0.0001. Neoadjuvant chemotherapy seemed to result in a relocation of Th1-committed CD4+ T cells from blood, presumably to the tumour, indicated by shifts in the methylation patterns, whereas no such shifts were seen for lineages corresponding to IL13, IL17A and FOXP3. CONCLUSION: Increased lineage commitment in CD4+ T cells, as determined by demethylation in predictive CpG sites, is associated with lower post-cystectomy tumour stage, complete response to neoadjuvant chemotherapy and overall better outcome, suggesting epigenetic profiling of CD4+ T cell lineages as a useful readout for clinical staging